DE2754770C2 - N-acyltetrapeptide amides, processes for their preparation and pharmaceuticals containing these compounds - Google Patents

Description

B methionyl, leucyl, norleucyl or norvalyl;
A methylene, ethylene or ethylidene;
E tert-butoxycarbonyl;

where the amino acid residues not mentioned separately have the L-configuration, but phenylglycyl D- or Has L-configuration and A has the meaning of ethylidene D-configuration, as well as their physiologically compatible Saize and usual complexes with prolonged effectiveness.

2. Process for the production of the peptides according to claim 1 and of the acid addition salts and complexes of these compounds, characterized in that tetrapeptides of the general formula II

H-Trp-B-Asp-Phg-NH ₂ (II)

wherein B has the meanings given in claim 1, with an (aminooxy) acyl group containing Compound of the following formula HI

E — N —O —A —C —X (HI)

I Il

HO

wherein E and A have the meanings given in claim 1 and

X stands for an activating group, the pivaloyloxy group or a group of the formula RO-CO ₂ -, in which R stands for a lower alkyl group, X also stands for a phenoxy group optionally substituted by nitro or halogen or for an N-succinimidoxy group, known per se Reacts manner and the compound obtained is optionally converted in a known manner into a physiologically acceptable acid addition salt or into a complex or from one of the salts a physiologically acceptable other salt or inner salt forms in a manner known per se or from a salt in a manner known per se the acid releases.

3. The method according to claim 2, characterized in that X is a hydroxyl group or a halogen atom means.

4. Medicament containing a compound according to claim 1 together with conventional carriers and / or Auxiliary materials.

The invention relates to phenylglycine-containing peptides with gastrin action, processes for their production and medicaments containing these compounds according to the claims.
The invention relates to N-acyltetrapeptide amides of the general formula I

E — N —O — A — C — Trp — B — Asp — Phg —NH ₂ (I)

I Il

HO

with the following meanings of B, A and E:
55

B methionyl, leucyl, norieucyl or norvalyl;
A methylene, ethylene or ethylidene;
E tert-butoxycarbonyl;

where the amino acid residues not mentioned separately have the L configuration, but phenylglycyl has D ^j or L configuration and A meaning ethylidene has D configuration, as well as their physiologically acceptable salts and customary complexes with prolonged effectiveness.

The phenylglycyl group denoted by Phg in the formula in each case means the L or D enantiomer.

The subject matter of the invention also includes the addition salts of the new peptides formed with acids and their complexes.

It is known that the part responsible for the biological effect, the active center, consists of 17 amino acids existing gastrins (the peptide hormone that stimulates gastric acid production)

oxylende located tetrapeptide amide
H-Trp-Met-Asp-Phe-NH ₂

whose acid production stimulating effect is about an order of magnitude less than that of the genes samthormons. Several processes are known for the synthesis of this tetrapeptide amide: J. C. Anderson et al .: J. Chem. Soc. C 108 (1967), Smirnow et al .: Himija prirodni sojedinjenij 7 94 (1971), M. Portelli, G. Renzi, G. E. Borin: Il Farmaco Ed. May be. 28, 322 (1973), J. M. Davey et al .: J. Chem. Soc. C 555 (I960), HU-PS 153 299, M. Dabrowska et al .: Roczniki Chemii 46 1295 (1972). Later more than a hundred were substituted derivatives and analogs of this tetrapeptide amide described (J. M. Davey et al .: J. Chem. Soc. C 555 (1966), J. S. Morley: Proc. Roy. Soc. 170B 97 (1968), Portelli and Renzi: Il Farmaco Ed. May be. 28 316 (1973), HU-PS153 375). Of these turned out the pentapeptide amide

BOC-jS-Ala-Trp-Met-Asp-Phe-NHj

(Pentagastrin) considered the most effective. It is commercially available under the name Peptavlon and is used for Determination of the maximum gastric acid production capacity used as a diagnostic tool.

Both of the above tetrapeptide amide as well as from numerous pentagastrin analogs were described in which individual amino acids are replaced by other: J. Morley: Proc. Roy. Soc. 170 B 97 (1968), E. Wünsch and K.-H. Deimer: Hoppe-Seylers Z. Physiol. Chem. 353 1246 (1972), Morley et al: J. Chem. Soc. C 522,726,910,809,715 (1968) Kenneru. a .: J. Chem. Soc. C 762 (1968), A. von Dungen and others: Liebigs Ann. Chem. 860 (1976), JA-PS 71 17234 and 16 743/1971. The effect of all these compounds is less than that of pentagastrin.

The peptides containing an a- (aminooxy) -carboxylic acid are resistant to numerous enzymes (L. Kisfaludy, M. Löw, T. Devenyi: Acta Biochem. Biophys. Acad. Sci. Hung. 6 393 (1971)). So is for example the long-term effects of the ACTH analogues containing an a- (aminooxy) -carboxylic acid at the amino end are likely due to the fact that the ar- (aminooxy) acyl group located at the amino end of the molecule protects against enzymes of the aminopeptidase type (HU-PS 167 655).

It has now been found that the pentagastrin analogs containing an (aminooxy) carboxylic acid at the amino end are very effective compounds. The (aminooxy) acetyl, α-D (aminooxy) propionyl and (aminooxy) propionyl derivatives are particularly effective. The effect of some of these derivatives exceeds that of pentagastrin.

According to the invention, the compounds of general formula (I) are prepared by adding tetrapeptide amides of the general formula (II)

35 H-Trp-B-Asp-Phg-NH ₂ (II)

wherein the meaning of B is the same as in formula (I), with a compound of the general formula (III) E-N-O-A-C-X (ffl)

HO

where E and A have the meaning given above,

X stands for an activating group, especially for hydroxyl, halogen, preferably chlorine, for the pivaloyloxy group or a group of the formula RO-CO ₂ -, where R is a lower alkyl group, preferably ethyl or isobutyl group, X furthermore for optionally by nitro or halogen-substituted phenoxy group, preferably p-nitro, 2,4,6-trichloro, 2,3,5-trichloro, pentachloro or pentafluorophenoxy group, or N-succinimidoxy group,

implements.

If X is hydroxyl, the acylation is preferably carried out with the aid of dicyclohexylcarbodiimide. If desired, the compound obtained becomes a physiologically acceptable acid addition salt or Complex implemented, or it becomes a physiologically compatible other salt or internal salt from one of its salts Salt is formed or the acid is released from one of its salts.

The reactive compound of the general formula

E-N-O-A-C-X

I Il

HO

is expediently a mixed anhydride or a mixed anhydride prepared with pivalic acid or a carbonic acid half ester active lister. Of the latter, the halogenated phenyl esters, especially the pentachloro and pentafluorophenyl esters, particularly preferred.

The tetrapeptide amides of the general formula used as starting material

H-Trp-B-Asp-Phg-NH ₂ (II)

are known per se by fragment condensation or by stepwise chain extension by means of the azide, the mixed anhydride, the dicyclohexylcarbodiimide and the active ester method or produced by means of other known activation processes

The functional groups not participating in the reaction are, if necessary, protected, in particular by groups that can be removed by means of hydrolysis, acidolysis or catalytic hydrogenation. The carboxyl group can, for example, be esterified with benzyl alcohol, tert-butanol or p-chlorobenzyl alcohol to be protected. The following groups can be used to protect the (aminooxy) and amino groups be: formyl, tosyl, trityl, trifluoroacetyl, o-nitrosulfenyl, phthalyl, p-chlorocarbobenzoxy, however, the carbobenzoxy and tert-butyloxycarbonyl groups are particularly preferred. The nitrogen atom on ίο The tryptophan's indole skeleton can be protected by a formyl group in some cases.

By an appropriate combination of the protective groups can be achieved that these by per se known methods (for example hydrolysis, acidolysis, catalytic hydrogenation) selectively or simultaneously are removable

According to a preferred embodiment of the process, the protected (aminooxy) acyl group is incorporated applied the pentahalophenyl ester method at the amino end; the active ester is with the carboxyl ends standing

H-Trp-B-Asp-Phg-NH ₂

brought to reaction. The synthesis of the latter is based on Z-L-Phg-OH or Z-D-Phg-OH (Z = protective group) and, after removing the protective group, builds with protected amino acid (N-hydroxysuccinimide) esters or - (pentahalogenphenyl) esters stepwise the tetrapeptide amide

H-Trp-B-Asp-D-Phg-NH ₂

H-Trp-B-Asp-L-Phg-NHj

on. The protective group Z is by treatment with glacial hydrogen bromide or by catalytic Hydrogenolysis removed. Instead of the protective group Z, the BOC group can also be used, which can be removed by milder acidic treatment.

The ester bond protecting the jö-cairboxyl group of aspartic acid can be in the dipeptide, tripeptide or tetrapeptide phase selectively or together with the group protecting the amino group by acidolysis, or by catalytic hydrogenolysis.

The end products can be obtained by crystallization, reprecipitation, column chromatography on silica gel or ion exchange chromatography cleaned with carboxymethyl cellulose.

The new compounds will be considered free, depending on the method used in making them Acids or obtained as salts. The salts formed with organic or inorganic acids can also be used known methods or converted to internal salts during cleaning. Preferred compounds according to the invention are:

2- (tert-BuiLyloxycarbonylaminooxy) -acetyl-L-tryptophyl-L-methionyl-L-asparagyl-D-phenylglycine amide _t 2- (tert-Bui: yIoxycarbonylaminooxy) -acetyI-L-tryptophyl-L-methionyl- L-asparagyl-L-phenylglycine-amide _t 2-D- (tert-butyloxycarbonyIaminooxy) -propionyI-L-tryptophyl-L-methionyl-L-asparagyl-D-phenylglycine amide,

2-D- (teR.-Butyloxycarbonylaminooxy) -propionyl-L-tryptophyl-L-methionyl-L-asparagyl-L-phenylglycine '· amide,

2- (tert-Bu1: yloxycarbonylaminooxy) -acetyl-L-tryptophyl-L-leucyl-L-asparagyl-D-phenylglycine amide, 2- (tert-But; yloxycarbonylaminooxy) -acetyl-L-tryptophyl-L-leucyl-L-asparagyl-L-phenylglycine amide, 2-D- (tert-ButyIoxycarbonylaminooxy) -propionyl-L-tryptophyl-L-leucyl-L-asparagyl-D-phenylglycine amide,

2-D- (tert-butyloxycarbonylaminooxy) -propionyl-L-tryptophyl-L-leucyl-L-asparagyI-L-phenylglycine amide,
2-D- (tert-butyloxycarbonylaminooxy) -propionyl-L-tryptophyl-L-norleucyl-L-asparagyl-D-phenylgIycinamide,

2-D- (tert-Elutyloxycarbonylaminooxy) -propionyl-L-tryptophyI-L-norvalalyI-L-asparagyl-D-phenylglycine amide.

The biological action of the new compounds according to the invention was determined using the method of Gosh and Schild (Br. J. Pharmac. Chemoth. 13,54 / 1958 /) investigated, the modification by Pissidis and Clark (Gut. 8,196 / 1967 /) was taken into account. As the animal experiments showed, the connections increase with intravenous Application significant acid production. What is particularly noteworthy is that the new compounds In contrast to pentagastrin it can also be absorbed in the small intestine and in some cases its acid secretion stimulatory effect at a dose of 20 µg / 100 g body weight is greater than that in one dose of 0.2 μg / 100 g of body weight intravenously administered pentagastrin, which also applies to enteral administration in larger doses does not stimulate acid production. It should also be emphasized that some derivatives in Absorption in the rectum exert a strong stimulating effect on acid secretion. This effect is in

in some cases greater than that of the pentagastrin used as a control. The level of effect both at Resorption in the small intestine as well as in the rectum enables a novel application of the compounds.

Table 1

The compounds applied enterally or parenterally to acid production increasing effect, expressed in% of the effect of 0.2 μ§ / 100 g i. v. Pentagastrin

Connection A

Phg

Application site and dose ^ g / 100 g)

Vein small intestine rectum

BOC-OGIy BOC-OGIy BOC-OGIy BOC-OGIy BOC-D-OAIa BOC-D-OAIa BOC-D-OAIa BOC-D-OAIa

Mead D. Leu D. Mead L. Leu L. Mead D. Leu D. Mead L. Leu L.

0.2 28

36

45

180

2.0
50

116

20th

20 µg

- 60 124 38 - 17th - 22nd 146 41 48 43

20th

25th

Physiologically compatible complexes are to be understood here as meaning compounds which are converted during the reaction of the peptides according to the invention with certain organic or inorganic compounds and ensure a retarded effect of the peptides. Organic compounds are, for example, certain gelatins, Carboxymethyl cellulose ^ alginic acid esters, poly (floretin phosphate), amino acid polymers and others Polymers and copolymers suitable. Of the inorganic compounds, the hydroxides come from certain Metals, for example zinc hydroxide, and sparingly soluble metal salts, for example phosphates and Pyrophosphates, in question. To achieve the retarding effect, certain silicates are also suitable, with the peptides also form insoluble complexes of a previously unexplained structure.

The peptides according to the invention and their salts and complexes can be used in pharmacy in the form of the usual Medicinal preparations are used. These preparations contain the compounds according to the invention accompanied by carriers suitable for enteral or parenteral administration. The drug preparations can be, for example, solid lyophilizates which, as carriers, do not react with the peptides Compounds such as carbohydrates. The compounds according to the invention can also for example, be formulated as dilute or concentrated suspensions or emulsions, which differ may contain indifferent preservatives and stabilizers.

The pharmaceutical preparations can be used as a diagnostic aid to determine the maximum gastric acid production can be used. Their application in therapy is indicated when the acid production capacity of the stomach is diminished or absent. This is the case with pre-atrophic gastritis and subacid complaints expect a good effect. Because the pentagastrin promotes the regeneration of the gastric mucosa Has trophic effect, the drug preparations are also used in atrophic processes the gastric mucosa favorable.

The method according to the invention is explained in more detail below on the basis of exemplary embodiments. The abbreviations used in the examples correspond to the abbreviations used in the specialist chemical literature (J. Biol Chem. 5 «247,977 / 1972 /). For ö - (aminooxy) acetic acid the symbol OGIy,
nooxy) propionic acid uses the symbol OAIa Further abbreviations:

DCC = dicyclohexyl carbodiimide

PCPOH = pentachlorophenol

PFPOH = pentafluorophenol

SuOH = N-hydroxysuccinimide

DMF = dimethylformamide

HMFA = hexamethylphosphoric acid triamide

60

The melting point of the compounds was determined using the Tottoli (Büchi) melting point measuring device. The thin-layer chromatograms were carried out on silica gel layers prepared according to Stahl (trade name: Silica gel G) added. The following solvent mixtures were used to develop the chromatograms used:

65

1. Chloroform

2. Chloroform

3. Chloroform

4. Ethyl acetate

5. Ethyl acetate

6. Ethyl acetate

7. Ethyl acetate

8. ethyl acetate;

9. Ethyl acetate:
10. Ethyl acetate:

Methanol methanol

η-hexane: acetic acid (pyridine: acetic acid (pyridine: acetic acid (Pyridine: acetic acid (pyridine: acetic acid (pyridine: acetic acid (pyridine: acetic acid (Pyridine: acetic acid

95

Water = 20 water = 20 water = 20 water = 20 water = 20 water = 20 Water = 20

H) 11) H) H) 11) H) H)

85

1: 1 1

Ninhydrin solution was used to develop the thin layer chromatograms. After spraying, the plates were ^{dried at 105 °} C. for about 5 minutes. The chromatograms were then placed in chlorine gas and, after ventilation, treated with o-tolidine-KJ solution.

For the column chromatographic purification of the substances silica gel (Kieseigel G) of a Korngrößebe · ¹ was rich of 62-200 μπι used.

example 1
a) N-benzyloxycarbonylJ-D-phenylglycine amide

24.0 g (84 mmol) of ZD-Phg-OH are dissolved in 170 ml of chloroform and 11.6 ml (84 mmol) of triethylamine are added to the solution. Subsequently, at - 1O ⁰ C was added dropwise 8.9 ml (84 mmol) Chlorkohlensäureäthylester. The solution of the mixed anhydride is carried out at - stirred for a further 10 minutes 10 ⁰ C, after removal of the cooling one hour long introduced ammonia gas. The reaction mixture is evaporated in vacuo the next day and the solid residue is isolated with water. The crude product is crystallized from ethanol. Aul "this manner are obtained 11.8 g (49.4%) chromatographically uniform ZD-Phg-NH _2, which melts at 171-173 ⁰ C R = 0.75;.? [A] _D = -76.8 ° (c = 1.0 in dimethylformamide).

Analysis for C | ₆ H, ₆ O ₃ N ₂ (M = 284.30): Calculated: C 67.59% H 5.67% N 9.85%; Found: C 67.43% H 5.70% N 9.98%.

b) D-phenylglycine amide hydrobromide

20.0 g (70 mmol) of ZD-Phg-NH ₂ (process stage a) are dissolved in 40 ml of acetic acid and 70 ml of 8 η acetic hydrogen bromide are added to the solution. After one hour, the reaction mixture is diluted with ether and the hydrobromide is filtered off. The salt containing a large excess of HBr is recrystallized from ten times the amount of ethanol. Melting point are obtained 14.15 g (87.5% 7 HD-Phg-NH ₂ · HBr: 263.0 to 263.5 ⁰ C (determined with the Koffler apparatus); R = 0.40; [a.? ] _D = -75.6 ° (c = 1.0; in dimethylformamide)

Analysis for C ₈ H ₁₀ ON ₂ Br (M = 231.10): Calculated: C 41.60%, H 4.77%, N 12.13%; Found: C 41.40% H 4.82% N 12.01%.

c) N-t-BeizyloxycarbonylVL-asparagyl-jß-ftert.-butylesterO-D-phenylglycine amide

6.31 g of Z-Asp (0'Bu) -OSu, 3.89 g (16.8 mmol) of HD-PHg-NH ₂ · HBr (example i, step b) and 2.110 ml (15 mmol) of triethylamine are suspended in 150 ml of chloroform and reacted with one another. The next day, the thick suspension is evaporated in vacuo and the residue is recrystallized from ethanol. 5.02 g (73.6%) of chromatographically uniform Z-Asp (O'Bu) -D-Phg-HN _{2 are obtained} , which melts at 179-181 ° C. A small sample is recrystallized from ethyl acetate and then melts at 188 -189 ° C. R? = 0.75; [a] _D = -59.5 ° (c = 1 in dimethylformamide).

Analysis Door C ₂₄ H ₂₉ O ₆ N ₃ (M = 455.50): Calculated: C 63.28% H 6.42% N 9.23%; Found: C 63.22% H 6.60% N 8.96%.

d) L-asparagyl-jS-itert.-butyl ester J-D-phenylglycine amide hydrochloride

12.83 g (28.2 mmol) of Z-Asp (O ^l Bu) -D-Phg-NH ₂ (example 1, stage c) are dissolved in 200 ml of DMF. While cooling, first 2.46 ml (28.2 mmol) of concentrated hydrochloric acid, then 2 g of palladium-on-charcoal are added to the solution. With stirring, hydrogen is passed through the suspension for 2.5 hours. The catalyst is then filtered off, the filtrate is evaporated and the residue is dissolved in ethanol. The solution is clarified that

Removed solvent in vacuo and solidified the residue with ether. 10.0 g (99%) of H-Asp (O'Bu) -D-Phg-NH ₂ · HCl, which ^{melts at 169-17O 0} C, are obtained. R? = 0.15.

e) N-itert.-ButyloxycarbonyO-L-methionyl-L-asparagyl-jS-itert.-butylesterO-D-phenylglycine amide

5.0 g (14 mmol) of H-ASp (O ¹ Bu) -D-PlIg-NHj · HCl (example 1, stage d) and 4.85 g (14 mmol) of BOC-Met-OSu are dissolved in 60 ml of dimethylformamide dissolved and added to the solution 1.96 ml (14 mmol) of triethylamine. The suspension is evaporated in vacuo the next day and the residue is isolated with water. 7.83 g of crude product are obtained. This is clarified hot in 40 ml of ethanol and precipitated at the boiling point by adding 40 ml of water. 6.57 g (84%) of chromatographically uniform BOC-Met-Asp (O'Bu) -D-Phg-NH ₂ , which melts at 194-196 ° C., are obtained. A small sample is recrystallized from ethanol and then melts at 197-198 ⁰ C. R? = 0.75; [a] _D = -67.2 ° (c = 1.0 in dimethylformamide).

Analysis for C ₂₆ H ₄₀ O ₇ N ₄ S (M = 552.69): Calculated: C 56.51% H 7.30% N 10.14% S 5.78%;
Found: C 56.81% H 7.34% N 9.98% S 5.69%.

0 L-methionyl-L-asparagyl-D-phenylglycine amide hydrochloride

5.6 g (10.1 mmol) of BOC-Met-Asp (O'Bu) -D-Phg-NH ₂ (example 1, step e) are dissolved in 10 ml of acetic acid and 3.5 ml (35 mmol) mercaptoethanol and 50 ml of glacial acetic acid 4 η HCl. After 30 minutes, the reaction mixture is evaporated in vacuo and the solid residue is isolated with anhydrous ether. 4.84 g of H-Met-Asp-D-Phg-NH ₂ · HCl (Rj = 0.1), which still contains excess hydrochloric acid, but is processed further without purification, are obtained.

g) N-itert.-ButyloxycarbonyO-L-tryptophyl-L-methionyl-L-asparagyl-D-phenylglycine amide

4.4 g (10.2 mmol) of H-Met-Asp-D-Phg-NH ₂ · HCl (Example 1, stage 0 and 4.10 g (10.2 mmol) of BOC-Trp-OSu are in 100 ml Dissolve dimethylformamide and add 2.85 ml (20.4 mmol) of triethylamine to the solution, evaporate the suspension in vacuo the next day and isolate the residue with water, giving 6.66 g (95.8%) of BOC-Trp -Met-Asp-D-Phg-NH ₂ , which melts with decomposition at 216-218 ° C. A small sample is recrystallized from 80% ethanol and then has a decomposition point of 222 ° C. R? = 0.3 ; [a) _D = -65.5 ° (c = 1.0 in dimethylformamide).

Analysis for C ₃₃ H ₄₂ O ₈ N ₆ S (M = 682.81):
Calculated: C 58.05% H 6.20% N 12.31% S 4.68%;
Found: C 58.23% H 6.30% N 12.60% S 4.86%.

h) L-tryptophyl-L-methionyl-L-asparagyl-D-phenylglycine amide

6.0 g (88.8 mmol) of BOC-Trp-Met-Asp-D-Phg-NH ₂ (Example 1, stage g) are treated with 60 ml of hydrochloric acid dioxane in the presence of 3.1 ml (44 mmol) of mercaptoethanol . After 15 minutes, the reaction mixture is diluted with anhydrous ether. 6.23 g of hydrochloride containing a large amount of excess hydrochloric acid are isolated by filtration. This is suspended in 150 ml of water, the pH of the suspension is adjusted to 7 with triethylamine, then stirred and the precipitate is filtered off and washed with water. This gives 4.0 g (78%) H-Trp-Met-Asp-D-Phg-NH _2, which melts with decomposition at 230-232 ⁰ C. R? = 0.3.

i) tert-Butyloxycarbonylaminooxy-acetic acid pentachlorophenyl ester

8.0 g (41.8 mmol) of BOC-OGIy-OH and 11.1 g (42.0 mmol) of pentachlorophenol are dissolved in 160 ml of anhydrous dioxane. The solution is cooled to at least 5 ° C. and then 8.6 g (42.0 mmol) of dicyclohexylcarbodiimide (DCC) are added. The reaction mixture is left to stand at room temperature overnight and then the precipitated dicyclohexylcarbamide (DCU) is filtered off. The filtrate is evaporated in vacuo and the solid residue is recrystallized from a mixture of dioxane and ethanol. In this way 13.7 g (74.3%) of BOC-OGIy-OPCP are obtained. Melting point: 167-168 ⁰ C. R? = 0.7.

Analysis door C ₃ Hi ₂ O ₅ NCl ₅ (M = 439.51):
Calculated: C 35.52% H 2.75% N 3.18% Cl 40.33%;
Found: C 35.37% H 2.90% N 3.31% Cl 40.12%.

j) 2- (tert-Butyloxycarbonylaminooxy) -acetyl-L-tryptophyl-L-methionyl-L-asparagyl-D-phenylglycine amide

1.50 g (2.57 niMoI) of H-Trp-Met-Asp-D-Phg-NH ₂ (example 1, stage h) are dissolved in 15 ml of dimethylformamide

suspended and 2.26 ml (5.14 mmol) of triethylamine and 1.20 g (2.73 mmol) of the suspension BOC-OGIy-OPCP (example 1, step 9) given. The suspension is stirred until everything has gone into solution is, and then left to stand overnight. After evaporation, the residue is solidified with ether. It 2.12 g of crude product are obtained, which is more than the theoretical yield. The raw product is in little of the solvent mixture 5 dissolved and applied to a column prepared from 20 g of silica gel. Is eluted with

'the solvent mixture 5 at a rate of 15 ml / h. The pure fractions are twice

ίο recrystallized from 20 ml of 80% ig; em ethanol. In this way, 0.82 g (41.4%)

; BOC-OGly-Trp-Met-Asp-D-Phg-NH ₂

obtained, which ^{melts at 199-200 0} C with decomposition. Rf = 0.15; Rf = 0.40; [a] _D = -40.3 ° (c = 1.0 in DMF).

Analysis for C _3j , H ₄₅ O ₁₀ N ₇ S (M = 755.86):

\ Calculated: C 55.62% H 6.00% N 12.97% S 4.24%;

Found: C 55.91% H 6.18% N 13.35% S 4.16%.

Example 2

; a) N-iBenzyloxycarbonyO-L-phenylglycine amide

27.6 g (97 mmol) of ZL-Phg-OH are dissolved in 300 ml of anhydrous tetrahydrofuran. To the solution, 13.55 ml (97 mmol) of triethylamine and thereafter at -10 ⁰ C 12.1 ml (97 mmol) of pivaloyl chloride. The suspension of the mixed anhydride is stirred for 10 minutes at -10 ⁰ C. After the cooling has been removed, ammonia gas is passed into the suspension for one hour. The next day, the reaction mixture is evaporated and the residue is worked up in a manner similar to that in the case of the D-enantiomer. 18.90 g (68.7%) of ZL-PhIg-NH ₂ , which melts at 172-173 ° C., are obtained. Rf = 0.75; [a] _D = + 80.7 ° (c = 1.0 in dimethylformamide).

ι b) L-phenylglycine amide hydrobromide

The procedure described in Example 1, process stage b, is used, but ZL-Phg-NHj is used as the starting material. The hydrobromide is obtained in 88.8% yield. Rf = 0.25; [a) _D = + 76.5 ° (c = 1.0 in DMF).

c) N-benzyloxycarbonyO-L-asparagyl-jiHtert.-butylester-L-phenylglycine-arnide

8.65 g (37.5 mmol) of HL-Phg-NH ₂ · HBr (Example 2, step b) are mixed with 21.6 g (37.5 mmol) of Z-Asp (O'Bu) -OPCP in 160 ml DMF reacted in the presence of 5.15 ml (37.5 mmol) of triethylamine. After stirring for 30 minutes, a further 5.15 ml (37.5 mmol) of triethylamine are added to the suspension. The next day the reaction mixture is evaporated in vacuo and the oily residue is isolated with ether. The precipitate is washed with water and the crude product (13.9 g) is recrystallized from aqueous ethanol. In this way, 10.6 g (65.8%) of chromatographically uniform Z-Asp (O'Bu) -L-Phg-NH ₂ , which melts at 207-208 ° C., are obtained. A small sample is urnkristallisiert from ethanol and melts then at 209 ⁰ C. Rf = 0.75; [a] _D = + 21.6 ° (c = 1.57 in DMF).

Analysis for C ₂₄ H ^ O ₆ N ₃ (M = 455.50)
Calculated: C 63.28% H 6.42% N 9.23%;
Found: C 63.50% H 6.38% N 9.23%.

The small amount of precipitate that precipitates out of the ethereal mother liquor of the crude product is filtered off, washed with water and recrystallized from ethanol. This gives 0.47 g (2.9%) of Z-Asp (O'Bu) -D-Phg-NH ₂ . Melting point: 188-189 ⁰ C; Rf = 0.75; [a] _D = -59.0 ° (c = 1 in DMF).

d) L-asparagyl-Xtert.-butyl esters ^ -L-phenylglycine amide hydrochloride

9.6 g (21 mmol) of Z-Asp (O'Bu) -L-Phg-NH ₂ (example 2, step c) are protected in the same way as for the D-diastereomer (example 1, step 4) removed. In this way, 8.03 g of chromatographically uniform H-Asp (O'Bu) -L-Phg-NH ₃ · HCl, which still contains excess hydrochloric acid, are obtained. Rf = 0.35.

e) N-iterL-BuryloxycarbonyO-L-methionyl-L-asparagyl - ^ - itert-butylesteri-L-phenylglycine amide

3.95 g (10.5 mmol) 1, -ASp (O ¹ Bu) -L-PlIg-NHj · HCl (Example 2, step d) and 5.22 g (12.6 mmol) BOC-Met-OPFP are dissolved in 50 ml of anhydrous dimethylformamide and 2.94 ml (21 mmol) of triethylamine are added to the solution. After one hour, the reaction mixture is evaporated in vacuo, the residue is isolated with η-hexane and washed with water. The 5.38 g of crude product are dissolved at the boil in 25 ml of methanol and precipitated with 25 ml of water.In this way, 4.70 g (81%)

BOC-Met-ASp (O ¹ Bu) -L-PlIg-NH ₂

obtain. A small sample of it is purified by falling over from a methanol-ether mixture and then melts at 162-163 ° C. R? = 0.75; [a] _D = -5.5 ° (c = 1.0 in dimethylformamide).

Analysis for C ₂₆ H ₄₀ O ₇ N ₄ S (M = 552.69):

Calculated: C 56.51% H 7.30% N 10.14% S 5.7%; Found: C 56.70% H 7.34% N 10.35% S 5.68%.

0 L-methionyl-L-asparagyi-L-phenylglycine-amide ^{hydrochloride?} ·

4.2 g (7.6 mmol) of BOC-Met-Asp (O ^l Bu) -L-Phg-NH ₂ are treated in the same way as the diastereomer. In this way 3.2 g (97%) of H-Met-Asp-L-Phg-NH ₂ · HCl, which melts at 198-200 ° C., are obtained. R? = 0.25 (compare the value of the D-diastereomer).

g) N-itert.-ButyloxycarbonylJ-L-tryptophyl-L-methionyl-L-asparagyl-L-phenylglycine amide

3.45 g (8 mmol) of Η-Met-Asp-L-Phg-NH ₂ · HCl (Example 2, step f) are prepared in a manner similar to the D- '

Diastereomer (Example 1, Step 7) coupled. In this way, 4.77 g (97.9%)

BOC-Trp-Met-Asp-L-Phg-NH ₂

obtained, which ^{melts at 191-193 0} C with decomposition. A small sample of it is recrystallized from 80% ethanol and then melts with decomposition at 200 ^° C. Rf = 0.35; [a] _D = 0 ° (c = 1.0 in DMF).

35 Analysis for C ₃₃ H ₄₂ O ₈ N ₆ S (M = 682.81):

Calculated: C 58.04% H 6.20% N 12.31% S 4.68%;

Found: C 58.05% H 6.21% N 12.64% S 4.77%.

40 h) N-Tryptophyl-L-methionyl-L-asparagyl-L-phenylglycine amide

One starts from 4.4 g (6.33 mmol) of BOC-Trp-Met-Asp-L-Phg-NH ₂ (example 2, stage g) and works in the manner described in example 1, step 8. 2.90 g (76.5%) of H-Trp-Met-Asp-L-Phg-NH ₂ are obtained, which ^{melts at 210 °} C. with decomposition. R? = 0.30.

i) 2- (tert-Butyloxycarbonylaminooxy) acetyl-L-tryptophyl-L-methionyl-L-asparagyl-L-phenylglycine amide

This is prepared in a similar manner to the D-diastereomer (Example 1, step j), using 1.0 g (1.71 mmol) of H-Trp-Met-Asp-L-Phg-NHj (Example 2, step 8) goes out. The crude product is purified on a silica gel column by eluting with the solvent mixture 10. 0.48 g (37.2%) of BOC-OGly-Trp-Met-Asp-L-Phg-NH _{2 is obtained} , which melts ^{at 169-17O 0 C with decomposition.} Rf = 0.35; [a] _D = + 8.2 ° (c = 1.0 in DMF).

55 Analysis for C ₃₅ H ₄₅ Oi ₀ N ₇ S (M = 755.86):

Calculated: C 55.62% H 6.00% N 12.97% S 4.24%;

Found: C 55.11% H 6.23% N 13.04% S 4.10%.

Example 3
a) 2- (tert-Butyloxycarbonylaminooxy) -D-propionic acid pentachlorophenyl ester

4.10 g (20 mmol) of BOC-D-OAIa-OH and 5.32 g (20 mmol) of pentachlorophenol are dissolved in 20 ml of dimethylformamide. The solution is cooled to 0 ⁰ C and treated with 4.12 g (20 mmol) of DCC. The reaction mixture is ^{stirred at 0 °} C. for half an hour and then left to stand at room temperature overnight. The precipitated DCU is filtered off and the filtrate is evaporated in vacuo. The residue is

stirred with anhydrous ether, and then filtered. The crude product obtained (6.25 g) is recrystallized from ethanol In this way, 5.80 g (64%) of BOC-D-OAIa-OPCP are obtained. Melting point: 123-124 ° C; R? = 0.85; U] = + 68.95 ° (c = 1.0 in dioxane).

Analysis for Ci ₄ H ₁₄ O ₅ NCIs (M = 453.54):

Calculated: C 37.10% H 3.10% Cl 39.10%;
Found: C 37.16% H 3.29% Cl 39.01%.

b) 2-D- (tert-Butyloxycarbonylaminooxy) -propionyl-L-tryptophyl-L-methionyl-L-asparagyl-D-

phenylglycine amide

1.50 g (2.57 mmol) of H-Trp-Met-Asp-D-Phg-NH ₂ (Example 1, stage h) are dissolved in 50 ml of dimethylformamide. 2.26 ml (5.14 mmol ) Triethylamine and 1.24 g (2.73 mmol) of BOC-D-OAIa-OPCP (Example 3, step 1) are given. After five hours, the reaction mixture is evaporated in vacuo and the residue is triturated with ether. The 1.90 g of crude product are recrystallized from 100 ml of 96% ethanol. There are obtained 1.06 g (53.5%) chromatographically uniform BOC-D-OAla-Trp-Met-Asp-D-Phg-NH ₂ , which melts at 200-204 ° C with decomposition R? = 0.15; R? = 0.30; [a] _D = -24.9 ° (c = 1.0 in dimethylformamide).

Analysis for C ₃₆ H ₄₇ Oi ₀ N ₇ S (M = 769.89):
Calculated: C 56.16% H 6.15% N 12.74% S 4.16%;
Found: C 56.33% H 5.88% N 12.70% S 4.09%.

Example 4

2-D- (tert-Butyloxycarbonyloxy) -propionyl-L-tryptophyl-L-methionyl-L-asparagyl-L-phenylglycine amide

1.0 g (1.71 mmol) of H-Trp-Met-Asp-L-Phg-NH ₂ (example 2, stage h) is dissolved in 30 ml of DMF. 0.24 ml (1.71 mmol ) Triethylamine and 0.85 g (1.88 mmol) of BOC-D-OAIa-OPCP (Example 3, step 1) are given. The reaction mixture is evaporated in vacuo the next day and the residue is isolated with anhydrous ether. The crude product (1.45 g) is dissolved in a small amount of the solvent mixture 10 and applied to a column prepared from 100 g of silica gel. Elution is carried out with the solvent mixture 10 and a rate of 15 ml / h. 0.74 g (56%) of BOC-D-OAIa-Trp-Met-Asp-L-Phg-NHj, which melts at 167-168 ° C., are obtained from the pure fractions. Rf = 0.30; Ia] ₀ = + 29.8 ° (<x = 1.0 in dimethylformamide).

Analysis for C ₃₅ H ₄₇ O ₁₀ N ₇ S (M = 769.89):
Calculated: C 56.16% H 6.15% N 12.74% S 4.16%;
Found: C 56.40% H 6.31% N 12.65% S 4.01%.

Example 5

a) N-iBenzyloxycarbonyO-L-leucyl-L-asparagyl-Xtert.-butylesterJ-D-phenylglycine amide

5.0 g (14.0 mmol) of H-Asp (O'Bu) -D-Phg-NH ₂ • HCl (Example 1, step d) and 5.13 g (14.2 mmol) of Z-Leu-OSu are dissolved in 60 ml of dimethylformamide and 1.96 ml (14.0 mmol) of triethylamine are added to the solution. The next day, the reaction mixture is evaporated in vacuo and the residue is isolated by trituration with water. The 7.32 g of crude product obtained are dissolved in ethanol, clarified and crystallized. 6.05 g (77.7%) of Z-Leu-ASp (O ¹ Bu) -D-PlIg-NH ₂ , which melts at 192-194 ° C., are obtained. Rf = 0.75. A small sample is recrystallized from ethanol and then melts at 195-196 ° C. [a] _D = -69.5 ° (c = 1.0 in DMF).

Analysis for C ₃₀ H ₄₀ O ₇ N ₄ (M = 568.67):

Calculated: C 63.36% H 7.09% N 9.8%;
Found: C 63.54% H 7.38% N 10.07%.

b) L-Leucyl-L-asparagyl-D-phenylglycine amide

5.20 g (9.15 mmol) of Z-Leu-Asp (O ^l Bu) -D-Phg-NH ₂ (example 5, stage a) are added for one hour with 50 ml

acetic acid η HBr implemented. The reaction mixture is evaporated in vacuo and with anhydrous

Ether isolates the hydrobromide. The 5.33 g of hydrobromide are suspended in 50 ml of chloroform. The pH value

the suspension is adjusted to 7 with triethylamine, then the gel-like product is filtered off and washed with ethanol. This gives 2.45 g (70.5%) H-Leu-Asp-D-Phg-NH _2, which melts at 218-220 ⁰ C. R? = 0.25.

c) N ^ terL-ButyloxycarbonyO-L-tryptophyl-L-methionyl-L-asparagyl-D-phenylglycine amide

2.25 g (5.93 mmol) of H-Leu-Asp-D-Phg-HN ₂ (Example 5, stage b) are dissolved in a mixture of 50 ml of DMF and 10 ml of HMFA and add 0.84 ml to the solution (5.93 mmol) of triethylamine and 2.65 g (6.6 mmol) of BOC-Trp-OSu were added. The reaction mixture becomes clear after stirring for four hours, it is left to stand overnight. The dimethylamide is then evaporated off in vacuo, the mixture that remains is diluted with water, the precipitate is left to stand cold and finally filtered off. The crude product (4.31 g) is boiled twice with ethyl acetate and then the suspension is filtered hot. This gives 2.87 g (72.8%) of BOC-Trp-Leu-Asp-D-Phg-NHz, melting at 195-200 ⁰ C R? = 0.55. The product is worked up without further purification.

d) L-Tryptophyl-L-leucyl-L-asparagyl-D-phenylglycine amide

2.8 g (4.2 mmol) of BOC-Trp-Leu-Asp-D-Phg-NH ₂ (Example 5, stage c) are in the presence of 0.59 ml (8.4 mmol) of mercaptoethanol with 30 ml of hydrochloric acid Treated with dioxane. After 15 minutes, the reaction mixture is diluted with anhydrous ether. The 2.60 g of hydrobromide are suspended in 150 ml of water, the pH of the suspension is adjusted to 7 with triethylamine, and the precipitate is filtered off and washed with water. In this way, 2.22 g (93.7%) of H-Trp-Leu-Asp-D-Phg-NH _{2 are obtained} , which melts at 246-248 ° C. with decomposition. R? = 0.35.

e) 2- (tert-Butyloxycarbonylaminooxy) acetyl-L-tryptophyl-L-leucyl-L-asparagyl-D-phenylglycine amide

.1.10 g (1.95 mmol) of H-Trp-Leu-Asp-D-Phg-NH ₂ Example 5, stage d) and 0.28 g (2.0 mmol) of triethylamine are dissolved in 10 ml of DMF and 0.92 g (2.10 mmol) of BOC-OGIy-OPCP (Example 1, step 9) were added to the solution. The next day the reaction mixture is evaporated in vacuo and the residue is triturated with anhydrous ether. The crude product (1.46 g) is filtered hot from ten times the amount of ethyl acetate. The 1.31 g of substance obtained are dissolved in a small amount of the solvent mixture 10 and applied to a column prepared from 80 g of silica gel. Elution is carried out with the solvent mixture 10 and a rate of 8 ml / h. From the pure fractions, 0.52 g of product is isolated, which is recrystallized from 80% ethanol. This gives 0.45 g (31.3%)

BOC-OGly-Trp-Leu-Asp-D-Phg-NH ₂

35 melting at 206-208 ⁰ C. Rf = 0.75; [a] _d = -51.7 ° (c = 0.5 in DMF).

Analysis for C ₃₆ H ₄₇ O ₁₀ N ₇ (M = 737.82):
Calculated: C 58.60% H 6.42% N 13.29%;
Found: C 58.70% H 6.65% N 13.51%.

Example 6
a) N-benzyloxycarbonyO-L-leucyl-L-asparagyHtert-butylesterJ-L-phenylglycine amide

The starting point is 4.0 g (11.2 mmol) of H-Asp (O'Bu) -L-Phg-NH ₂ · HCl (example 2, stage d) and the procedure given in example 5, step 1, is used Way. The crude product (5.42 g) is clarified from ethanol. 3.0 g (47%) of Z-Leu-Asp (O'Bu) -L-Phg-HN ₂ , which melts at 193-194 ° C. with decomposition, are obtained. Rj = 0.70, R? = 0.65. [a] _D = 0 ° (c = 1.0; in dimethylformamide).

Analysis for C ₃₀ H ₄₀ O ₇ N ₄ (M = 568.67):
Calculated: C 63.36% H 7.09% N 9.85%;
Found: C 63.79% H 7.30% N 9.59%.

b) L-Leucyl-L-asparagyl-jß-tert-butylesterJ-L-phenylglycine amide hydrochloride

2.75 g (4.84 mmol) of Z-Leu-Asp (O'Bu) -L-Phg-NH ₂ (Example 6, stage a) are dissolved in 50 ml of DMF and in the presence of 0.45 ml (5th , 2 mmol) of concentrated hydrochloric acid with 0.5 g of palladium activated carbon as a catalyst by passing hydrogen through it. After two hours, the catalyst is filtered off and the filtrate is evaporated in vacuo. The residue is isolated with anhydrous ether. 2.25 g (98.5%) of H-Leu-Asp (O'Bu) -L-Phg-NH ₂ · HCl, which melts at 182-183 ° C., are obtained. R? = 0.15.

c) L-Leucyl-L-asparagyl-L-phenylglycine amide hydrochloride 2.25 g (4.80 mmol) H-Leu-Asp (O ^l Bu) -L-Phg-NH ₂ · HCl (Example 6, Stage b) are made more hydrochloric with 20 ml

Acetic acid reacted. After 40 minutes, the reaction mixture is evaporated in vacuo and the residue is triturated with ether. In this way 2.0 g (99%) H-Leu-Asp-L-Phg-NH ₂ - HCI obtained, which melts at 21O ⁰ C with decomposition R? = 0.20.

d) N-itert.-Butyloxycarbonyy-L-tryptophyl-L-methionyl-L-asparagyl-L-phenylglycine amide

2.2 g (5.3 mmol) of H-Leu-Asp-L-Phg-NH ₂ · HCl (Example 6, stage c) are dissolved in 50 ml of DMF in the presence of 1.48 ml (10.6 mmol) of triethylamine reacted with 2.32 g (5.8 mmol) of BOC-Trp-OSu. The reaction mixture is evaporated in vacuo and the residue is triturated with water. The crude product (3.30 g) is dissolved in 70 ml of 80% ethanol, clarified and then crystallized. 2.30 g (65%) are obtained

BOC-Trp ~ Leu-Asp-L-Phg-NH ₂
that melts at 205-207 ⁰ C with decomposition R ^ = 0.4Q-. [d [ _D = -7.3 ° (c = 1.0 in dimethylformamide).

e) L-Tryptnphyl-L-leucyl-L-asparagyl-L-phenylglycine amide

2.23 g (3.25 mmol) of BOC-Trp-Leu-Asp-L-Phg-NH ₂ (Example 6, stage d) are in the presence of 0.47 ml (6.7 mmol) of mercaptoethanol with 25 ml of hydrochloric acid Dioxane implemented. After 10 minutes the reaction mixture is diluted with anhydrous ether. The hydrochloride is separated off and suspended in water, the pH of the suspension is adjusted to 7 with triethylamine and the precipitate is filtered off and washed with water. In this way, 1.16 g (61%) of H-Trp-Leu-Asp-L-Phg-NH ^ are obtained at 214-215 ° C. with decomposition

melts. R? = 0.40.

0 2- (tert-butyloxycarbonylaminooxy) acetyl-L-tryptophyl-L-leucyl-L-asparagyl-L-phenylglycine amide

0.57 g (1.0 mmol) of H-Trp-Leu-Asp-L-Phg-NH ₂ (example 6, step e) and 0.48 g (1.1 mmol) of BOC-OGIy-OPCP (example 1 , Step 9) are dissolved in 15 ml of DMF and 0.14 ml (1.0 mmol) of triethylamine are added to the solution. The next day the reaction mixture is evaporated in vacuo and the residue is isolated with ether. The 0.81 g of crude product obtained are purified in the manner already described for the D-diastereomer. 0.30 g (40.5%) of BOC-OGly-Trp-Leu-Asp-L-Phg-NH ₂ are obtained.

The product melts with decomposition at 196-198 ⁰ C. R ^ = 0.35. [a] _D = 0 ° (c = 1.0 in dimethylformamide).

Analysis for C ₃₆ H ₄₇ O ₁₀ N ₇ (M = 737.82):
Calculated: C 58.60% H 6.42% N 13.29%;
Found: C 58.35% H 6.20% N 13.07%.

Example 7
2-D- (tert-butyloxycarbonylaminooxy) -propiony! -L-tryptophyl-L-leucyI-L-asparagyl-D-phenylglycine amide

1.15 g (2.03 mmol) of H-Trp-Leu-Asp-D-Phg-NH ₂ (Example 5, step d) and 1.0 g (2.2 mmol) of BOC-D-OAIa-OPCP ( Example 3, step 1) are reacted in 10 ml of dimethylformamide. The reaction mixture contains 0.29 ml (2.03 mmol) of triethylamine. The next day the reaction mixture is evaporated in vacuo and the residue is isolated with ether. The crude product (1.56 g) is dissolved in 50 ml of 80% ethanol containing 0.3 ml of acetic acid. The solution is clarified before crystallizing. 0.86 g (56%) of BOC-D-OAla-Trp-Leu-Asp-D-Phg-NHj, which melts at 213-214 ° C. with decomposition, are obtained. Rj = 0.2; R? = 0.4. [a] _D = -28.7 ° (c = 1.0 in DMF).

Analysis for C ₃₇ H ₄₉ O ₁₀ N ₇ (M = 751.85):
Calculated: C 59.11% H 6.57% N 13.04%;
Found: C 59.10% H 6.52% N 13.20%.

Example 8
60

2-D- (tert-Butyloxycarbonylaminooxy) -propionyl-L-Tryptophyl-L-Ieucyl-L-asparagyl-L-phenylglycine amide

0.57 g (1.0 mmol) of H-Trp-Leu-Asp-L-Phg-NH ₂ (example 6, step e) and 0.50 g (1.1 mmol) of BOC-OAIa-OPCP (example 3 , Step 1) are reacted in 15 ml of DMF in the presence of 0.14 ml (1.0 mmol) of triethylamine. The reaction mixture is evaporated in vacuo the next day, the residue is triturated with ether and the crude product (0.77 g) is dissolved in a small amount of the solvent mixture 10 and applied to a column prepared from 30 g of silica gel. Elution is carried out with the solvent mixture 10 at a rate of 5 ml / h. 0.46 g of product are isolated from the pure fractions. This is in

10 ml of methanol dissolved and precipitated by adding 70 ml of ether. 0.26 g (34.2%) are obtained

BOC-D-OAIa-Trp-Leu-Asp-L-Phg-NHj '

which melts at 184-185 ° C with decomposition. Rf = 0.4. [a] _D = + 18.8 ° (c = 1.0; dimethylformamide). S '

Analysis for C ₃₇ H ₄₉ Oi ₀ N ₇ (M = 751.85):
Calculated: C 59.11% H 6.57% N 13.04%;
Found: C 58.92% H 6.40% N 12.88%.

Example 9 f

h a) N-tert-butyloxycarbonyl-1-norleucine pentafluorophenyl ester g

10.0 g (24.3 mmol) of BOC-NIe-OH · DCHA are in a mixture of 75 ml of ether and 25 ml of 2 η Schwe- ί

Feisäure shaken until everything has gone into solution. After the phases have separated, the organic *

Phase with 25 ml of 2 η sulfuric acid and then extracted with water. The ethereal solution is evaporated in vacuo. The oily product (5.44 g) is dissolved together with 4.42 g (24 mmol) of pentafluorophenol in 30 ml of ethyl acetate. The solution is cooled to 0 ⁰ C and treated with 4.63 g (22.5 mmol) of dicyclohexylcarbodiimide. The suspension is left in the cold for an hour and then filtered. The filtrate is evaporated in vacuo, the residue is dissolved in 50 ml of η-hexane and the solution is extracted by shaking with 5 × 20 ml of 5% NaHCOi solution, then with 2 × 20 ml of water. The solution is dried and then evaporated in vacuo. The oily residue solidifies in the cold. This gives 8.12 g (91%) of BOC-Nle-OPFP, which melts at 55-57 ⁰ C. [a] _D = -26.8 ° (c = 1.0 in dioxane); R? = 0.80; R? = 0.85.

Analysis door C ₁₇ H ₂₀ O ₄ NF ₅ (M = 397.35):
Calculated: C 51.39% H 5.07% F 23.91%;
Found: C 51.51% H 4.68% F 24.66%.

b) L-asparagyl (tert-butyl ester) -D-phenylglycine amide

10.6 g (23.2 mmol) of Z-Asp (O'Bu) -D-Phg-NH ₂ (example 1, stage c) are suspended in 350 ml of methanol and in the presence of 1.1 g of 10% palladium activated carbon reduced with stirring with hydrogen from atmospheric pressure. After 3 hours, the suspension is filtered, the catalyst is washed out with DMF and the washing liquid is combined with the filtrate. After evaporation in vacuo, the solid residue is made from 100 ml; Recrystallized methanol. In this manner, 5.5 g (74.0%) H-Asp (O ^l Bu) -D-Phg-NH ₂ are obtained, which melts at 170-171 ⁰ C. R? = 0.15.

40 Analysis for C ₆ H ₂₁ O ₄ N ₃ (M = 321.28):

Calculated: C 59.79% H 7.21% N 13.08%;

Found: C 59.80%, H 7.10%, N 13.18%.

c) N-tert-butyloxycarbonyl-L-norvalyl-L-asparagyl-jS-Ktert-butylesteri-D-phenylglycine amide

1.60 g (5.0 mmol) of H-Asp (O ^l Bu) -D-Phg-NH ₂ and 1.99 g (5.0 mmol) of BOC-NIe-OPFP are dissolved in 10 ml of DMF in the presence of 0 , 70 ml (5.0 mmol) of triethylamine reacted. The next day, the reaction mixture is evaporated in vacuo, the solid residue is taken up in ether and filtered off. 2.45 g (91.9%) of BOC-Nle-Asp (O ^l Bu) -D-Phg-NH ₂ , which ^{melts at 207-208 5} ° C., are obtained. R? = 0.75; [a] _D = -62.8 ° (c = 1.0, in dimethylformamide). :

Analysis for C ₂₇ H ₄₂ O ₇ N ₄ (M = 534.64):

Calculated: C 60.65% H 7.92% N 10.48%; Found: C 60.63% H 8.04% N 10.18%.

d) L-norleucyl-L-asparagyl-D-phenylglycine amide hydrochloride

2.20 g (4.11 mmol) of BOC-NIe-Asp (O'Bu) -D-Phg-NH ₂ are treated with 15 ml of 4 η hydrochloric acid acetic acid. After one hour, the reaction mixture is evaporated in vacuo, the solid residue becomes filtered with ether 1.70 g (99%) of H-NIe-Asp-D-Phg-NH ₂ · HCl, which melts at 225 ° C. with decomposition, are obtained. R / = 0.20, [a \ _D = -63.7 ° (c = 1.0 in dimethylformamide).

e) N-tert-butyloxycarbonyl-L-tryptophyl-L-norleucyl-L-asparagyl-D-phenylglycine amide

1.57 g (3.78 mmol) of H-NIe-Asp-D-Phg-NH ₂ · HCl and 1.52 g (3.78 mmol) of BOC-Trp-OSu are in

50 ml of DMF reacted in the presence of 1.06 ml (7.6 mmol) of triethylamine. The reaction mixture is on evaporated the next day in vacuo and the residue filtered with water. 2.45 g (97.5%) are obtained

BOC-Trp-Nle-Asp-D-Phg-NH ₂ , which melts at 217 ° C with decomposition. Rf = 0.35, [ff] _D = -32.7 ° (c = 1.0; in dimethylformamide).

Analysis for C ₃₄ H ₄₄ O ₈ N ₆ (M = 664.77):
ίο Calculated: C 61.43% H 6.67% N 12.64%;
Found: C 61.41% H 6.86% N 12.65%.

f) L-tryptophyl-L-norleucyl-L-asparagyl-D-phenylglycine amide hydrochloride
15th

2.20 g (3.32 mmol) of BOC-Trp-Nle-Asp-D-Phg-NH ₂ are treated with 25 ml of 4 η hydrochloric acid dioxane in the presence of 1.20 ml (17 mmol) of mercaptoethanol. After 15 minutes the solution is evaporated in vacuo and the residue is solidified with ether. After filtering, 1.99 g (99.0%) of H-Trp-Nle-Asp-D-Phg-NH ₂ · HCl, which melts at 217-221 ° C. with decomposition, are obtained. R? = 0.35 [a) _D = -54.2 ° (c = 1.0; in dimethylformamide).

g) 2-D- (tert-Butyloxycarbonylaminooxy) -propionyl-L-tryptophyl-L-norleucyl-L-asparagyl-D-

phenylglycine amide

1.80 g (3.0 mmol) of H-Trp-Nle-Asp-D-Phg-NH ₂ · HCl and 1.36 g (3.0 mmol) of BOC-DO AIa-OPCP are in the presence of 1.26 ml (9.0 mmol) of triethylamine reacted in 30 ml of DMF. The next day, the reaction mixture is evaporated in vacuo and the residue is filtered with water. The substance is purified by chromatography. This gives 0.99 g of BOC-D-OAla-Trp-Nle-Asp-D-Phg-NHj, which melts at 211-212 ° C. with decomposition. R? = 0.35; Rj = 0.25. [a] _D = -22.8 ° (c = 1.0; in dimethylformamide).

Analysis for C ₃₇ H ₄₉ O ₁₀ N ₇ (M = 751.85):
Calculated: C 59.11% H 6.57% N 13.04%;
Found: C 59.10% H 6.30% N 13.21%.

Example 10
a) N-tert-butyloxycarbonyl-L-norvaline pentafluorophenyl ester

8.0 g (20 mmol) of BOC-Nva-OH · DCHA are shaken with 60 ml of ether and 20 ml of 2 η sulfuric acid until everything has dissolved. The ethereal solution is washed with 20 ml of 2N sulfuric acid, dried and evaporated in vacuo. The remaining oil is dissolved together with 3.70 g (20 mmol) of pentachlorophenol in 25 ml of ethyl acetate. The solution is ^{reacted at 0} ° C. with 3.92 g (19 mmol) DCC for one hour. The suspension is then filtered, the filtrate is evaporated in vacuo and the residue is dissolved in 50 ml of n-hexane. The solution is washed with 5 × 20 ml of 5% NaHCO ₃ solution and then with 2 × 20 ml of water. The solution is evaporated in vacuo. The oily residue crystallizes in the cold. This gives 6.21 g (81%) of BOC-Nva-OPFP, which melts at 60-62 ⁰ C. R? = 0.70, [a] _D = -32.3 ° (c = 1.0 in dioxane).

so analysis for C ₁₆ H ₁₈ O ₄ NF ₅ :

Calculated: C 50.14% H 4.73% F 24.78%;
Found: C 50.06% H 4.64% F 24.63%.

b) N-tert-butyloxycarbonyl-L-norvalyl-L-asparagyl-jS-itert.-butyl ester O-D-phenylglycine amide

1.60 g (5.0 mmoles) of H-Asp (O'Bu) -D-Phg-NH ₂ (Example 9, step b) and 1.91 g (5.0 mmoles) of BOC-Nva-OPFP

are reacted in 10 ml of DMF in the presence of 0.70 ml (5 mmol) of triethylamine

The reaction mixture was evaporated in vacuo and the residue was filtered with ether. 2.36 g (87.4%) are obtained

BOC-NVa-ASp- (O ¹ Bu) -D-Ph ₈ -NH ₂ , which ^{melts at 200-202 0} C R? = 0.75; [a] _D = -66.7 ° (c = 1.0 in dimethylformamide).

Analysis for C ₂₆ H ₄₀ O ₇ N ₄ (M = 520.61):

Calculated: C 59.98% H 7.74% N 10.76%;
Found: C 60.08% H 7.68% N 10.96%.

c) L-norvalyl-L-asparagyl-D-phenylglycine amide hydrochloride

2.0 g (3.84 mmol) of BOC-Nva-Asp (O ^l Bu) -D-Phg-NH ₂ are treated with 15 ml of 4 η hydrochloric acid acetic acid. After one hour the suspension is diluted with ether and filtered cold. 1.52 g (98%) of I-Nva-Asp-D-Phg-NFV HCl are obtained. The hydrochloride melts under Zerzetzung at 204 ⁰ C. R? = 0.15. 5 [a] p = -58.0 ° (c = 1.0 in dimethylformamide).

d) N-tert-butyloxycarbonyl-L-tryptophyl-L-norvalyl-L-asparagyl-D-phenylglycine amide

1.50 g (3.74 mmol) of H-Nva-Asp-D-Phg-NH ₂ · HCl and 1.50 g (3.74 mmol) of BOC-Trp-OSu are dissolved in 30 ml of dimethylformamide in the presence of 1, 05 ml (7.5 mmol) of triethylamine reacted. The next day, the reaction mixture is evaporated in vacuo and the residue is filtered with water. The crude product (2.36 g) is recrystallized from 300 ml of 80% ethanol. 1.54 g (66.2%) are obtained

BOC-Trp-Nva-Asp-D-Phg-NHz '
which melts at 240 ⁰ C with decomposition. Rj = 0.35. [a] _D = -60.5 ° (c = 1.0; in dimethylformamide).

Analysis for C ₃₃ H ₄₂ O ₈ N ₆ (M = 650.71): 20

Calculated: C 60.91% H 6.51% N 12.92%;
Found: C 60.66% H 6.59% N 12.57%.

e) L-Tryptophyl-L-norvalyl-L-asparagyl-D-phenylglycine amide hydrochloride 25

1.50 g (2.30 mmol) of BOC-Trp-Nva-Asp-D-Phg-NH ₂ are treated with 20 ml of 4 η hydrochloric acid dioxane, 0.77 ml (11 mmol) of mercaptoethanol being added. After 30 minutes the suspension is evaporated in vacuo and the solid residue is filtered with ether. In this way 1.34 g (99.0%) of H-Trp-Nva-Asp-D-Phg-NH ₂ · HCl are obtained, which melts at 216 ° C. with decomposition. R? = 0.30. 30 [a] _D = -53.3 ° (c = 1.0; in dimethylformamide).

0 2-D- (tert-butyloxycarbonylaminooxy) propionyl-L-tryptophyl-L-norvalyl-L-asparagyl-D-phenylglycine amide

1.24 g (2.11 mmol) of H-Trp-Nva-Asp-D-Phg-NH ₂ · HCl and 0.95 g (2.10 mmol) of BOC-D-OAIa-OPCP are dissolved in 40 ml of DMF in The presence of 0.88 ml (6.3 mmol) of triethylamine reacted. The next day the reaction mixture is evaporated and the residue is filtered with water, then washed with water and ether. The crude product (1.40 g) is recrystallized in a mixture of pyridine, acetic acid and water in a ratio of 20: 6: 11. This gives 0.72 g of BOC-D-Trp-Nva-Oala-Asp-D-Phg-NH _2, which melts at 210 ⁰ C 40th R ^ = 0.28; [a] _D = -26.5 ° (c = 1.0; in dimethylformamide)

Analysis for C ₃₆ H ₄₇ O ₁₀ N ₇ (M = 737.79):
Calculated: C 58.61% H 6.42% N 13.29%;
Found: C 58.57% H 6.20% N 13.17%. 45

Claims (1)

Patent claims: L N-acyltetrapeptide amides of the general formula I.
E — N — O — A — C — Trp — B — Asp — Phg — NH ₂ (I) I Il HO with the following meanings of B, A and E: